Effectiveness in woody biomass utilization is highly dependent on its genetics and physiology. We performed morpho-anatomical, chemical, and biomethane productivity characterizations of one-year-old woody stems in three shrub Salix viminalis genotypes: a diploid (Energo) and its two autotetraploid derivatives (PP-E7 and PP-E13). Tetraploidization affected changes in stem morpho-anatomy and corresponding improved chemical features and biomethane productivity, considerably more pronounced in tetraploid PP-E13, while PP-E7 was more similar to diploid Energo. Compared to diploid Energo, in tetraploid PP-E13 morphometric analysis showed increased stem diameter and higher wood fiber radial double wall thickness, while microscopic analysis suggested higher syringyl to guaiacyl (S:G) ratio of the wood fiber cell wall. Presented changes in stem morpho-anatomy of tetraploid PP-E13 compared to diploid Energo correspond to the improved chemical features: the lower Klason lignin content and higher S:G ratio, the higher cellulose and xylan content, and lower cellulose crystallinity (Crl). Presented improved chemical features, along with the increase in ash content, resulted in a 7.3

Successful use of woody species in reducing climatic and environmental risks of energy shortage and spreading pollution requires deeper understanding of the physiological functions controlling biomass productivity and phytoremediation efficiency. Targets in the breeding of energy willow include the size and the functionality of the root system. For the combination of polyploidy and heterosis, we have generated triploid hybrids (THs) of energy willow by crossing autotetraploid willow plants with leading cultivars (Tordis and Inger). These novel Salix genotypes (TH3/12, TH17/17, TH21/2) have provided a unique experimental material for characterization of Mid-Parent Heterosis (MPH) in various root traits. Using a root phenotyping platform, we detected heterosis (TH3/12: MPH 43.99%; TH21/2: MPH 26.93%) in the size of the root system in soil. Triploid heterosis was also recorded in the fresh root weights, but it was less pronounced (MPH%: 9.63–19.31). In agreement with root growth characteristics in soil, the TH3/12 hybrids showed considerable heterosis (MPH: 70.08%) under in vitro conditions. Confocal microscopy-based imaging and quantitative analysis of root parenchyma cells at the division–elongation transition zone showed increased average cell diameter as a sign of cellular heterosis in plants from TH17/17 and TH21/2 triploid lines. Analysis of the hormonal background revealed that the auxin level was seven times higher than the total cytokinin contents in root tips of parental Tordis plants. In triploid hybrids, the auxin–cytokinin ratios were considerably reduced in TH3/12 and TH17/17 roots. In particular, the contents of cytokinin precursor, such as isopentenyl adenosine monophosphate, were elevated in all three triploid hybrids. Heterosis was also recorded in the amounts of active gibberellin precursor, GA19, in roots of TH3/12 plants. The presented experimental findings highlight the physiological basics of triploid heterosis in energy willow roots.

Hybrid vigor and polyploidy are genetic events widely utilized to increase the productivity of crops. Given that bioenergy usage needs to be expanded, we investigated triploid hybrid vigor in terms of the biology of biomass-related willow traits and their relevance to the control of biomethane production. To produce triploid hybrid genotypes, we crossed two female diploid Swedish cultivars (Inger, Tordis) with two male autotetraploid willow (Salix viminalis) variants (PP-E7, PP-E15). Field studies at two locations and in two successive years recorded considerable midparent heterosis (MPH%) in early shoot length that ranged between 11.14 and 68.85% and in the growth rate between 34.12 and 97.18%. The three triploid hybrids (THs) developed larger leaves than their parental cultivars, and the MPH% for their CO2 assimilation rate varied between 0.84 and 25.30%. The impact of hybrid vigor on the concentrations of plant hormones in these TH genotypes reflected essentially different hormonal statuses that depended preferentially on maternal parents. Hybrid vigor was evinced by an elevated concentration of jasmonic acid in shoot meristems of all the three THs (MPH:29.73; 67.08; 91.91%). Heterosis in auxin-type hormones, such as indole-3-acetic acid (MPH:207.49%), phenylacetic acid (MPH:223.51%), and salicylic acid (MPH:27.72%) and benzoic acid (MPH:85.75%), was detectable in the shoots of TH21/2 plants. These hormones also accumulated in their maternal Inger plants. Heterosis in cytokinin-type hormones characterized the shoots of TH3/12 and TH17/17 genotypes having Tordis as their maternal parent. Unexpectedly, we detected abscisic acid as a positive factor in the growth of TH17/17 plants with negative MPH percentages in stomatal conductance and a lower CO2 assimilation rate. During anaerobic digestion, wood raw materials from the triploid willow hybrids that provided positive MPH% in biomethane yield (6.38 and 27.87%) showed negative MPH in their acid detergent lignin contents (from -8.01 to -14.36%). Altogether, these insights into controlling factors of above-ground growth parameters of willow genotypes support the utilization of triploid hybrid vigor in willow breeding to expand the cultivation of short rotation energy trees for renewable energy production.

Previously, studies on RING-type E3 ubiquitin ligases in cereals were preferentially focused on GW2 genes primarily controlling seed parameters in rice and wheat. Here we report cloning two HvYrg genes from barley that share significant homology with rice GW2 gene. In antisense genotypes efficiency of gene silencing varied between genes and transgenic lines: ASHvYrg1: 30–50% and ASHvYrg2: 20–27%. Reduced activity of both genes altered shoot system with increasing number of side shoots. Changes in leaf width, weight, or plant weight and height reached significant levels in some transgenic lines. Lowering expression of the two barley HvYrg genes caused opposite responses in spike development. Plants with ASHvYrg1 gene construct showed earlier heading time and prolonged grain-filling period, while plants from ASHvYrg2 genotype flowered in delay. Digital imaging of root development revealed that down-regulation of HvYrg1 gene variant stimulated root growth, while ASHvYrg2 plants developed reduced root system. Comparison of seed parameters indicated an increase in thousand grain weight accompanied with longer and wider seed morphology. In summary we conclude that in contrast to inhibition of GW2 genes in rice and wheat plants, down-regulation of the barely HvYrg genes caused substantial changes in vegetative organs in addition to alteration of seed parameters.

Soil salinity can limit the use of marginal lands for biomass production based on cultivation of short-rotation woody crops as energy willow. Here, we compare salt stress responses of the diploid, productive cultivar (Energo), and its artificially produced autotetraploid (PPE-2; PPE-7; PPE-13) variants. After pre-testing the effects of various salt concentrations, willow plants with different genome sizes were exposed to 1.5 g NaCl kg(-1) soil (electrical conductivity (EC) value: 7.04 mS/cm). Digital imaging of shoot surface area (green pixel) and root surface (white pixel) indicated variable improvements in growth responses of tetraploids relative to diploid ones in saline soils. After nine weeks of salt stress, increase in salt adaptation capability of tetraploid plants was indicated by larger biomass, leaf and root weights under salinity (1.5 g NaCl kg(-1) soil) relative to diploids. Biomass weights were significantly higher in the case of tetraploid PPE-2 plants with increased water consumption and leaf water content than of diploid plants. The inhibitory effect of salt stress on photosynthetic assimilation rates was less significant in plants with doubled genome. The Na+ accumulation was reduced in leaves of tetraploids and increased in their roots, while the K+ ion content was higher in their leaves than in diploids. Tetraploidy improved K+/Na+ ratio in leaves and roots of willow plants under normal soil condition. This parameter was less reduced in tetraploid leaves exposed to salt stress. The described tetraploid energy willow genotypes with salt tolerance can play a role in the extended use of green energy.

Biomass productivity of shrub willow plants grown in short rotation system can be improved by genetic means or by innovative cropping technologies. In the present study we analyzed the growth and physiological responses of willow plants to plant biostimulators, such as 1-triacontanol (TRIA), a saturated primary alcohol and seaweed extract (Kelpak(center dot)). Testing a novel approach, we soaked stem cuttings in TRIA or Kelpak solutions for 48 h before plantation. These treatments enlarged height and diameter of stems, furthermore increased stem and leaf weights in comparison to the water control. In agreement with these greenhouse observations, field tests showed statistically significant enhancement in height and diameter of woody stems harvested in winter. Application of 25% Kelpak solution was the most effective in stimulating all traits including stem weight per plant after pre planting treatment of cuttings. In an alternative treatment protocol, Kelpak was applied as foliar treatment or in combination between TRIA (10 mg L-1) treatment of cuttings and foliar spray with Kelpak 1 or 2% solutions in the greenhouse. The green pixel numbers revealed variable degrees of stimulation in shoot growth. These treatments resulted in an increase in stem and leaf weights. Improvement of photosynthetic functions was indicated by more efficient electron transport rates (ETRs) of photosystems. An increased nicotinamide and thiamine contents were detected in the leaves of stimulated plants. The present study can serve as a foundation for additional laboratory and field studies optimizing the application of these stimulators in energy plantations.

The biomass productivity of the energy willow Salix viminalis as a short-rotation woody crop depends on organ structure and functions that are under the control of genome size. Colchicine treatment of axillary buds resulted in a set of autotetraploid S. viminalis var. Energo genotypes (polyploid Energo [PP-E]; 2n = 4x = 76) with variation in the green pixel-based shoot surface area. In cases where increased shoot biomass was observed, it was primarily derived from larger leaf size and wider stem diameter. Autotetraploidy slowed primary growth and increased shoot diameter (a parameter of secondary growth). The duplicated genome size enlarged bark and wood layers in twigs sampled in the field. The PP-E plants developed wider leaves with thicker midrib and enlarged palisade parenchyma cells. Autotetraploid leaves contained significantly increased amounts of active gibberellins, cytokinins, salicylic acid, and jasmonate compared with diploid individuals. Greater net photosynthetic CO2 uptake was detected in leaves of PP-E plants with increased chlorophyll and carotenoid contents. Improved photosynthetic functions in tetraploids were also shown by more efficient electron transport rates of photosystems I and II. Autotetraploidization increased the biomass of the root system of PP-E plants relative to diploids. Sections of tetraploid roots showed thickening with enlarged cortex cells. Elevated amounts of indole acetic acid, active cytokinins, active gibberellin, and salicylic acid were detected in the root tips of these plants. The presented variation in traits of tetraploid willow genotypes provides a basis to use autopolyploidization as a chromosome engineering technique to alter the organ development of energy plants in order to improve biomass productivity.

1 1 2 3 DUPLICATED GENOME REPROGRAMS ENERGY WILLOW GROWTH 4 5 6 7 8 Corresponding author: Dénes Dudits 9 Institute of Plant Biology 10 Biological Research Center, Hungarian Academy of Sciences 11 6726 Szeged, Temesvári kr. 62 Hungary 12 Tel.: 36 62 599-600 13 dudits.denes@brc.mta.hu 14 15 16 17 Research Area: Genes, Development and Evolution 18 19 20 21 Plant Physiology Preview. Published on January 4, 2016, as DOI:10.1104/pp.15.01679

Genetic improvement of complex traits such as drought adaptation can be advanced by the combination of genomic and phenomic approaches. Semi-robotic phenotyping platform was used for computer-controlled watering, digital and thermal imaging of barley plants grown in greenhouse. The tested barley variants showed 0-76% reduction in green pixel-based shoot surface area in soil with 20% water content, compared to well-watered plants grown in soil with 60% water content. The barley 'HvA1' gene encoding the group 3 LEA (Late Embryogenesis Abundant) protein exhibited four (A-D) haplotypes as identified by the EcoTILLING and subsequent DNA sequencing. The green pixel mean value of genotypes with haplotype D was higher than the mean value of the remaining haplotypes, indicating a pivotal role of haplotype D in optimizing the green biomass production under drought condition. In water limitation, the canopy temperature of a highly sensitive genotype was 18.0 degreesC, as opposed to 16.9 degreesC of leaves from a tolerant genotype as measured by thermal imaging. Drought-induced changes in leaf temperature showed moderate correlation with the water use efficiency (r2 = 0.431). The haplotype/trait association analysis based on the t-test has revealed a positive effect of a haplotype B (SNPs:GCCCCTGC) in a gene encoding the barley fungal pathogen induced mRNA for pathogen-related protein ('HvPPRPX'), on harvest index, thousand grain weight, water use efficiency and grain yield. The presented pilot study established a basic methodology for the integrated use of phenotyping and haplotyping data in characterization of genotype-dependent drought responses in barley.

Jelenleg vilagszerte es hazankban is a szarazsagstressz az egyik legjelentősebb termes korlatozo tenyező a gabonafelek, igy az arpa eseteben is. Kovetkezeskeppen a legfőbb nemesitesi celok koze tartozik uj, fokozottabb szarazsagtoleranciaval rendelkező fajtak előallitasa. A nemesites sikeressege nagymertekben fugg attol, hogy sikerul-e megertenunk e komplex es kvantitativ jellegű tulajdonsag kialakitasaban reszt vevő genetikai faktorokat. A komplex tulajdonsagok, mint peldaul a szarazsaghoz tortenő alkalmazkodas fejleszteset elősegitheti a genomikai es fenomikai megkozelitesek kombinacioja. A meglevő termeszetes genetikai variabilitas meghatarozasa pedig ertekes informacioval szolgalhat a szarazsagtűresben resztvevő genek funkciojat illetően. A genotipus-fenotipus asszociaciok egyuttes vizsgalata lehetőve teheti a vizsgalt genjeloltek szarazsagtoleranciaban betoltott funkciojanak megerősiteset. Az arpa szarazsagtűresben potencialisan szerepet jatszo genjeloltek genomikai adatait a polimorfizmusok hatekony feltarasara alkalmas EcoTILLING technologia hasznalata utjan nyertuk. A fenomikai adatsorokat uveghazi korulmenyek kozott, Komplex Stressz Diagnosztikai Rendszer alkalmazasaval allitottuk elő. Az EcoTILLING modszer egy nagy kapacitasu, viszonylag alacsony koltseggel kivitelezhető eljaras, mely lehetőve teszi a termeszetes populaciokban megtalalhato polimorfizmusok hatekony detektalasat. A modszer a TILLING (Targeting Induced Local Lesions IN Genomes) technologia egyik valtozata, amely bizonyos PCR lepeseken alapszik, ugymint heteroduplex kezes, valamint a kialakult mismatch poziciok nukleaz enzimekkel tortenő emesztese. A technologia alkalmazasa utjan egyarant lehetőve valik a polimorfizmusok feltarasa, illetve a haplotipusok elkulonitese az azonositott egyedi haplotipusok szekvenalasa utjan. Kutatasaink celjai kozott szerepelt az arpa szarazsagtűresenek hattereben allo genetikai faktorok vizsgalata a fenomikai es genomikai megkozelitesek egyesitesenek utjan. Az EcoTILLING reakcio magaban foglalta a vizsgalt genszakasz fluoreszcens nukleotidok jelenleteben vegzett PCR-amplifikaciojat. A kapott amplikonokat a heteroduplex kepzes utan az egyszalu DNS-re specifikus aktivitassal rendelkező Cel1-es endonukleazzal emesztettuk a „mismatch” poziciokban, majd ABI 377-es szekvenalo keszuleken kulonitettuk el a fragmentumokat molekulameretuk szerint. Vegul az azonositott haplotipusokat forward es reverz iranybol is megszekvenaltuk. Vizsgalatainkhoz a vilag szamos pontjarol gyűjtott 96 genotipust /termesztett fajtakat, okotipusokat es vad valtozatokat/ is tartalmazo, szarazsagtűres szempontjabol variabilis arpa kollekciot allitottunk ossze. A genjeloltek kivalasztasa soran a szarazsagtűressel foglalkozo szakirodalmi adatokra (gen expresszios, illetve QTL terkepezesi vizsgalatok, transzgenikus kutatasok) tamaszkodtunk. Elsődleges celunk volt az arpa szarazsag tűreseben potencialisan szerepet jatszo genjeloltek termeszetes genetikai variabilitasanak feltarasa az EcoTILLING technologia, mint a polimorfizmusok azonositasara alkalmas eszkoz alkalmazasa utjan. Tovabbi celjaink kozott szerepelt konnyen detektalhato genetikai markerek (potencialisan „genen beluli markerek”) kifejlesztese az atfedő haplotipus szekvenciak birtokaban, amelyek lehetőve teszik a főbb, elsősorban aminosav szinten kulonbseget mutato haplotipusok elkuloniteset. Az alkalmazott technologia segitsegevel mintegy 1,5 millio bazisparnyi szekvencia vizsgalata nyoman 94 egyedi allelvarianst kulonitettunk el a 9 genre tervezett 18 amplikon elemzese utjan. Egy bazisparnyi elterest (SNP) 185, inszercio/delecio-t (InDel) pedig 46 esetben azonositottunk, a polimorfizmusok atlagos gyakorisaga 1 SNP/92 bp illetve 1 InDel/372 bp volt. Az amplikonok vizsgalata soran az azonositott haplotipusok szama 2 es 9 koze esett. Osszesen 4 gen – Hordeum vulgare L. AR-h gene for aldose reductase (HvARH1), Hordeum vulgare L. HVA1 gene (HvA1), Hordeum vulgare L. HvSRG6 gene for stress responsive gene protein 6 (HvSRG6), Hordeum vulgare L. AP2 transcriptional activator gene (HvDRF1) - eseteben olyan informativ polimorfizmusokat konvertaltunk at genetikai markerekke, melyek altal elkulonithetők a valoszinűsithetően funkcionalis allelvariansok. Tovabba ezek a konnyen detektalhato genetikai markerek hasznosithatok kapcsoltsagi terkepezesek soran illetve a markerekre alapozott szelekcioban. Kutatasaink tovabbi fontos eleme volt egy arpa genotipus torzskollekcio szarazsagra adott valaszreakcioinak jellemzese a morfologiai, fiziologiai es agronomiai parameterek monitorozasa utjan kontroll es stresszelt korulmenyek kozott Komplex Stressz Diagnosztikai Rendszer hasznalataval. Celul tűztuk ki tovabba a tesztelt genotipusok fenotipikus stressz parameterek es a szarazsagtűresben szerepet jatszo genjeloltek azonositott haplotipusainak osszetetele kozotti kapcsolatok feltarasat. Celunk volt ezen tulmenően egy, az arpa szarazsagtűrasenek jellemzesere hasznalhato alapvető metodika kialakitasa a fenotipusos es haplotipusos eredmenyek egyuttes kiertekelese utjan. A szarazsagtolerancia mertekenek es tovabbi fontos agronomiai tulajdonsagok Komplex Stressz Diagnosztikai Rendszerben tortenő tesztelese erdekeben osszeallitottunk egy 23 genotipust tartalmazo arpa torzskollekciot. A morfologiai es fiziologiai tulajdonsagok monitorozasat kontroll (60 %-os vizellatottsag a teljes tenyeszidő alatt) es stressz (20 %) korulmenyek kozott kulonboző kepalkoto eljarasok alkalmazasaval vegeztuk. A morfologiai, fiziologiai tulajdonsagok, illetve a szarazsagtűressel osszefuggesbe hozhato agronomiai parameterek vizsgalatat tobbek kozott digitalis fenykepezes, illetve hőkameras levelhőmerseklet meghatarozas alkalmazasaval egy fel-automata fenotipizalasra alkalmas eszkoz, a Komplex Stressz Diagnosztikai Rendszer hasznalataval vegeztuk el. Vizsgalataink soran jelentős mertekű korrelaciot talaltunk a vizsgalt genotipusok termesmennyisege illetve tovabbi fontos agronomiai parameterei kozott, mint peldaul a harvest index, a vizhasznosito kepesseg es az ezerszemsuly. Ezen eredmenyek a Komplex Stressz Diagnosztikai Rendszer hasznalatabol fakado előnyokre hivhatjak fel a figyelmet. A hőkameras meresek alkalmaval a szarazsag stressznek kitett novenyek eseteben magasabb levelhőmersekleti ertekeket detektaltunk. A kivalasztott genotipusok eseteben a mert levelhőmerseklet es a pixel alapu zoldfelulet kozott kapcsolatot lehetett kimutatni. A meghatarozott pixel alapu zoldfelulet alapjan a genotipusokat stressz reakciojuk szerint tolerans es szenzitiv kategoriakba soroltuk az csokkenes 45 %-os erteket elkulonitő szintkent valasztva. Az analizis soran a tolerans es a szenzitiv kategoriakba tartozo genotipusok haplotipus osszeteteleben jelentős kulonbsegeket talaltunk a HvA1 gen eseteben. A genotipusokat termesstabilitasuk szerinti rangsor alapjan szinten tolerans es a szenzitiv kategoriakba soroltuk a termeskieses 55 %-os erteket kuszobszintkent veve alapul. A tolerans, illetve szenzitiv genotipusok haplotipus osszetetelet osszehasonlitva a HvDRF1 es a HvNHX1 gen eseteben lenyeges eltereseket talaltunk. A haplotipusok es a fenotipusos parameterek t-teszten alapulo asszociacios vizsgalata soran az arpa patogen gombak altal indukalt feherjejet kodolo gen (HvPPRPX) B-haplotipusa valamint a harvest index, az ezerszemsuly, a vizhasznositasi hatekonysag es a szemtermes kozott pozitiv osszefuggest tartunk fel. A kiserleteink eredmenyekent rendelkezesre allo fenotipus es haplotipus informaciokat tartalmazo adatsorok fontos kiindulasi pontjai lehetnek a szarazsagtűresben fontos szereppel rendelkező genvariansok QTL illetve asszociacios vizsgalatokkal tortenő azonositasa soran.

In the present study, allele mining was conducted on a panel of drought related candidate genes in a set of 96 barley genotypes using EcoTILLING, which is a variant of the targeting induced local lesions in genomes (TILLING) technology. Analyzing approximately 1.5 million basepairs in barley a total number of 94 verified unique haplotypes were identified in 18 amplicons designed for 9 genes. Overall, 185 single nucleotide polymorphisms (SNPs) and 46 insertions/deletions (INDELs) were detected with a mean of 1SNP/92 bp and 1INDEL/372 bp genomic sequence. Based on overlapping haplotype sequences, markers were developed for four candidate genes (HvARH1, HvSRG6, HvDRF1, HVA1), which allows distinguishing between the main haplotypes showing either differences in amino acid sequence or which have larger INDELs in the promoter region. As "proof of concept", the HvARH1 and HvSRG6 haplotypes were tested for the level of abscisic acid-induced gene expression in subsets of genotypes belonging to different haplotype categories. An integrated database was developed to contain information about the genes, genotypes, and haplotypes analyzed in this study. The database supplies profound information about the natural variation in the tested drought related candidate genes providing a significant asset for further mapping studies dealing with this highly polygenic trait.

When the primary amino groups of α-chymotrypsin were modified with acetic, propionic, succinic, citraconic and phthalic acid anhydrides, the modifications enhanced the stability of the enzyme in 60% aqueous solutions of 1,4-dioxane, ethanol and acetonitrile. The acetylation of the amino groups resulted in the lowest stabilization, but the citraconylated, propionylated and succinylated forms of α-chymotrypsin exhibited increased stabilities in all the aqueous organic solvents studied. The modification of α-chymotrypsin with phthalic anhydride was accompanied by very extensive activation. The near-UV circular dichroism spectroscopic measurements did not indicate significant structural changes in the acylated forms of α-chymotrypsin, with the exception of the phthalic anhydride-modified enzyme. The spectral changes in the phthalic anhydride-modified enzyme suggest enhanced interactions between the aromatic chromophores and alterations in the disulfide contribution. The improvement in stability may be related to the modifications caused by the reorientation and the increased interactions of the aromatic side-chains, particularly in the case of the phthalic anhydride derivative.